Cloning and expression of the luxAB genes and the entire luxCDABE cassette from different luminescent organisms (Vibrio fischeri, V. harveyi, and Photorhabdus luminescens) has led to the widespread and expanding application of the bacterial lux system as a reporter of gene expression and regulation (Liu, et al., Plasmid, 44:250-261, 2000), as well as a sensor of environmental pollutants and metabolic functions in a wide range of prokaryotic organisms (Applegate et al., Appl. Environ. Microbiol., 64:2730-2735, 1998 and Sayler, G. S., and Ripp, S., Current Opinion in Biotechnology, 11:286-289, 2000).
Bacterial luciferase expressed from luxAB genes catalyzes the oxidation of reduced riboflavin 5′-phosphate (FMNH2) and a long-chain aliphatic aldehyde (tetradecanal) synthesized by luxCDE genes, yielding FMN (flavin mononucleotide), fatty acid, water, and greenish blue light. The cofactor, FMNH2, is provided by the flavin oxidoreductase enzyme (NADPH-FMN Oxidoreductase or FMN oxidoreductase) in prokaryotes. Engineering schemes using only the luxAB genes require the addition of exogenous aldehyde substrate, typically n-decylaldehyde, to generate a bioluminescent response. Use of the entire luxCDABE operon, however, allows for intrinsic whole-cell bioluminescence without the requirement for exogenous addition of chemicals or co-factors. Thus, the bioreporter remains completely self-sufficient in its ability to produce visible light in response to specific chemicals or physical agents. Consequently, the luxCDABE system has found unusual applications as remote, real-time, reagentless components in bioelectronic devices, whole cell logic gates for biocomputing, in situ functional imaging and analysis of recombinant strain released to the environment and in vivo imaging of the course of systemic infection in animal hosts
While both luxAB and luc (firefly luciferase) have been used as reporters of gene expression in eukaryotic cells; a reagentless real-time bioreporter system independent of an exogenous substrate or an excitation source such as needed for GFP, has not been available for eukaryotic applications in research, medicine or biotechnology. In contrast to gene expression in bacterial hosts where the lux cassette is transcribed as a polycistronic mRNA, eukaryotic systems generally require a separate promoter preceding each gene. This stringent requirement of gene expression has limited the application of bacterial lux genes in eukaryotic organisms solely to luxAB derivatives. Fused luxAB genes have been constructed, allowing for the expression of luciferase under a single promoter in eukaryotic hosts including Saccharomyces cerevisiae, mammalian, plant, and insect cells, as well as in vitro in reticulocyte lysates. In these fusions, the carboxyl terminal of the α subunit of the luciferase is linked by a short polypeptide ranging from 1-22 amino acids to the amino terminal of the β subunit by eliminating the stop codons of the α subunit. Although luxAB fusions can generate bioluminescence when supplemented with a requisite aldehyde substrate, relative levels of activity vary widely depending on the expression system, growth assay, and availability of the cofactor, FMNH2. For example, luminescence levels not more than 2000 times above background were detected during constitutive expression of a fused luxAB construct under the control of a PGK promoter in S. cerevisiae. 
In addition to luxAB fusions, attempts have also been made to simultaneously express luxA and luxB separately from a dual promoter expression system. Successful expression and assembly of the V. harveyi luciferase protein subunits into a functional dimeric form has been demonstrated in plant protoplasts, transformed calli, and leaves of transformed plants (Koncz et al., Proceedings of the National Academy of Sciences of the United States of America, 84:131-135, 1987). Although the independently expressed subunits remain stable, protein folding kinetics upon fusion are significantly altered as a function of temperature, which proves especially detrimental in eukaryotic systems (Escher et al., Molecular and Cellular Biology, 13:4860-4874, 1989). Moreover, generation of in vivo bioluminescence in eukaryotic cells is difficult because the availability of the cofactor, FMNH2, is limited for the bioluminescent reaction (Meighen, E. A., Microbiological Reviews, 55:123-142, 1991). Therefore, direct measurement of bacterial luciferase activity in eukaryotes without disruption of the cell membrane and loss of cell viability has yet to be achieved.
The use of the prokaryotic lux-based bioluminescent reporter system for transcriptional fusions has revolutionized both applied and basic research capabilities by allowing for real-time, reagentless monitoring of a wide variety of extracellular analytes and intracellular genetic events. However, the same technology has not been available for eukaryotic applications. Although both GFP and Luc reporter proteins are commonly used in eukaryotic systems, both are subject to external manipulations (exogenous light excitations or luciferin additions) prior to quantitation and cannot be exploited in reagentless, real-time, on-line bioassays.
Reporter proteins including bacterial luciferase (LuxAB), β-galactosidase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP) and firefly luciferase (Luc) have been widely used as indicators of gene expression and regulation as well as sensors of metabolic functions in both prokaryotic and eukaryotic systems (Greer and Szalay Luminescence 17:43-74, 2002). However, the requirement of an exogenous substrate or excitation source in these reporter assays has restricted their use primarily to laboratory and in vitro applications. Accordingly, there exists a need for a self-sustaining bioluminescent system in eukaryotic cells.